The recent isolation of several key broad neutralizing antibodies (bnAb) from HIV infected subjects have set the stage for the development of novel strategies for the induction of similar classes of vaccine induced neutralizing antibody responses. Among these recent bnAb isolates, both 10E8 and DH511 target the distal MPER epitopes on HIV Envelope (Env) gp41. Structural and functional analysis of DH511 lineage antibodies have shown that distal MPER epitope bnAbs are among the most broad and potent HIV neutralizing antibodies, thus establishing them as important for vaccine design. For immunogen design, we will partner with the Scripps team in Project 1, and use our new computational program, Antigen Receptor Mutation Analyzer for Detection of Low Likelihood Occurrences (ARMADiLLO), that allows for definition of the critical antibody somatic mutations to be induced, to determine the key IAs that a successful vaccine will need to target. The use of KI mouse models bearing BCR of BnAbs and their UCAs (Unmutated Common Ancestor) has been a major advance that will allow the optimization of unmutated precusor-targeting sequential immunogens, and allow the course of antibody affinity maturation of candidate immunogens to be followed. We will use the newly isolated DH511 distal MPER bnAb clonal lineage UCA, intermediate antibodies (IAs) and bnAbs as reagents upon which to design sequential immunogens and to test these immunogens in a powerful, new knock-in mouse model of bnAb development developed by the Fred Alt laboratory in the Animal Models Core. Moreover, recent breakthoughs in understanding Rhesus macaque immunobiology has allowed similar HIV neutralizing antibody genealogies to be defined and followed throughout vaccination regimens. Our specific Aims are: Aim 2.1. Define functional improbable intermediate antibody (IA) somatic mutations that a successful vaccine will need to select to lead to bnAb induction. Aim 2.2. Define immunogens derived from Project 1 that activate DH511 unmutated precursors and intermediate antibodies in small animal models and in Rhesus macaques. The studies in Project 2 will synergize with studies in Project 1 and Small Animal Models Core to lead to novel designs of immunogens that will lead to initiation and selection of affinity matured distal MPER bnAbs.